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Essentials Glycoprotein VI (GPVI) binds collagen, starting thrombogenesis, and fibrin, stabilizing thrombi.GPVI‐dimers, not monomers, recognize immobilized fibrinogen and fibrin through their D‐domains.Collagen, D‐fragment and D‐dimer may share a common or proximate binding site(s) on GPVI‐dimer.GPVI‐dimer–fibrin interaction supports spreading, activation and adhesion involving αIIbβ3. SummaryBackgroundPlatelet collagen receptor Glycoprotein VI (GPVI) binds collagen, initiating thrombogenesis, and stabilizes thrombi by binding fibrin.ObjectivesTo determine if GPVI‐dimer, GPVI‐monomer, or both bind to fibrinogen substrates, and which region common to these substrates contains the interaction site.MethodsRecombinant GPVI monomeric extracellular domain (GPVI ex) or dimeric Fc‐fusion protein (GPVI‐Fc2) binding to immobilized fibrinogen derivatives was measured by ELISA, including competition assays involving collagenous substrates and fibrinogen derivatives. Flow adhesion was performed with normal or Glanzmann thrombasthenic (GT) platelets over immobilized fibrinogen, with or without anti‐GPVI‐dimer or anti‐αIIbβ3.ResultsUnder static conditions, GPVI ex did not bind to any fibrinogen substrate. GPVI‐Fc2 exhibited specific, saturable binding to both D‐fragment and D‐dimer, which was inhibited by mFab‐F (anti‐GPVI‐dimer), but showed low binding to fibrinogen and fibrin under our conditions. GPVI‐Fc2 binding to D‐fragment or D‐dimer was abrogated by collagen type III, Horm collagen or CRP‐XL (crosslinked collagen‐related peptide), suggesting proximity between the D‐domain and collagen binding sites on GPVI‐dimer. Under low shear, adhesion of normal platelets to D‐fragment, D‐dimer, fibrinogen and fibrin was inhibited by mFab‐F (inhibitor of GPVI‐dimer) and abolished by Eptifibatide (inhibitor of αIIbβ3), suggesting that both receptors contribute to thrombus formation on these substrates, but αIIbβ3 makes a greater contribution. Notably, thrombasthenic platelets showed limited adhesion to fibrinogen substrates under flow, which was further reduced by mFab‐F, supporting some independent GPVI‐dimer involvement in this interaction.ConclusionOnly dimeric GPVI interacts with fibrinogen D‐domain, at a site proximate to its collagen binding site, to support platelet adhesion/activation/aggregate formation on immobilized fibrinogen and polymerized fibrin.

Frailty is associated with non-prescription of anticoagulants, independently of CHA DS -VASc and HAS-BLED. It could be an important unmeasured factor in anticoagulation decisions. The utility of explicit frailty measurements in anticoagulation decisions and patient outcomes requires further research. Geriatr Gerontol Int 2017; 17: 2178-2183.

Analysis of oxylipins by liquid chromatography mass spectrometry (LC-MS) is challenging because of the small mass range occupied by this diverse lipid class, the presence of numerous structural isomers, and their low abundance in biological samples. Although highly sensitive LC-MS/MS methods are commonly used, further separation is achievable by using drift tube ion mobility coupled with high-resolution mass spectrometry (DTIM-MS). Herein, we present a combined analytical and computational method for the identification of oxylipins and fatty acids. We use a reversed-phase LC-DTIM-MS workflow able to profile and quantify (based on chromatographic peak area) the oxylipin and fatty acid content of biological samples while simultaneously acquiring full scan and product ion spectra. The information regarding accurate mass, collision-cross section values in nitrogen (DT CCSN2) and retention times of the species found are compared to an internal library of lipid standards as well as the LIPID MAPS Structure Database by using specifically developed processing tools. Features detected within the DT CCSN2 and m/z ranges of the analyzed standards are flagged as oxylipin-like species, which can be further characterized using drift time alignment of product and precursor ions distinctive of DTIM-MS. This not only helps identification by reducing the number of annotations from LIPID MAPS, but also guides discovery studies of potentially novel species. Testing the methodology on Salmonella enterica serovar Typhimurium infected murine bone-marrow derived macrophages and thrombin activated human platelets yields results in agreement with literature. This workflow has also annotated features as potentially novel oxylipins, confirming its ability in providing further insights into lipid analysis of biological samples.

BackgroundPlatelets are essential to physiological hemostasis or pathological thrombus formation. Current antiplatelet agents inhibit platelet aggregation but leave patients at risk of systemic side-effects such as hemorrhage. Newer therapeutic strategies could involve targeting this cascade earlier during platelet adhesion or activation via inhibitory effects on specific glycoproteins, the thrombogenic collagen receptors found on the platelet surface.AimsGlycoprotein VI (GPVI) is increasingly being recognized as the main platelet-collagen receptor involved in arterial thrombosis. This review summarizes the crucial role GPVI plays in ischemic stroke as well as the current strategies used to attempt to inhibit its activity.Summary of reviewIn this review, we discuss the normal hemostatic process, and the role GPVI plays at sites of atherosclerotic plaque rupture. We discuss how the unique structure of GPVI allows for its interaction with collagen and creates downstream signaling that leads to thrombus formation. We summarize the current strategies used to inhibit GPVI activity and how this could translate to a clinically viable entity that may compete with current antiplatelet therapy.ConclusionFrom animal models, it is clear that GPVI inhibition leads to an abolished platelet response to collagen and reduced platelet aggregation, culminating in smaller arterial thrombi. There is now an increasing body of evidence that these findings can be translated into the development of a bleeding free pharmacological entity specific to sites of plaque rupture in humans.

Objectives Platelet activation underpins thrombus formation in ischemic stroke. The active, dimeric form of platelet receptor glycoprotein (GP) VI plays key roles by binding platelet ligands collagen and fibrin, leading to platelet activation. We investigated whether patients presenting with stroke expressed more GPVI on their platelet surface and had more active circulating platelets as measured by platelet P-selectin exposure. Methods 129 ischemic or hemorrhagic stroke patients were recruited within 8h of symptom onset. Whole blood was analyzed for platelet-surface expression of total GPVI, GPVI-dimer, and P-selectin by flow cytometry at admission and day-90 post-stroke. Results were compared against a healthy control population (n = 301). Results The platelets of stroke patients expressed significantly higher total GPVI and GPVI-dimer (P<0.0001) as well as demonstrating higher resting P-selectin exposure (P<0.0001), a measure of platelet activity, compared to the control group, suggesting increased circulating platelet activation. GPVI-dimer expression was strongly correlated circulating platelet activation [r2 = 0.88, P<0.0001] in stroke patients. Furthermore, higher platelet surface GPVI expression was associated with increased stroke severity at admission. At day-90 post-stroke, GPVI-dimer expression and was further raised compared to the level at admission (P<0.0001) despite anti-thrombotic therapy. All ischemic stroke subtypes and hemorrhagic strokes expressed significantly higher GPVI-dimer compared to controls (P<0.0001). Conclusions Stroke patients express more GPVI-dimer on their platelet surface at presentation, lasting at least until day-90 post-stroke. Small molecule GPVI-dimer inhibitors are currently in development and the results of this study validate that GPVI-dimer as an anti-thrombotic target in ischemic stroke.

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